3.1 Monoclonal Antibodies
The technique for producing monoclonal antibodies was pioneered in the mid-1970's when Kohler and Milstein successfully fused spleen lymphocytes with malignant cells (myelomas) of bone marrow primary tumors [C. Milstein, Sci. Am. 243(4): 66-74 (1980)]. The method created a hybrid cell line, arising from a single fused cell hybrid, or clone, which possessed characteristics of both the lymphocytes and myeloma cell lines. Like the lymphocytes (taken from animals primed with sheep red blood cells as antigens), the fused hybrids, called hybridomas, secreted a single type of immunoglobulin specific to the antigen; moreover, like the myeloma cell lines, the hybrid cell lines were immortal. The combination of these two features has had a major impact in fields of research and medicine in which conventional antisera are used. Whereas antisera derived from vaccinated animals are variable mixtures of antibodies which never can be reproduced identically, monoclonal antibodies are highly specific immunoglobulins of a single type. The single type of immunoglobulin secreted by a hybridoma is specific to one and only one antigenic determinant on the antigen, a complex molecule having a multiplicity of antigenic determinants. For instance, if the antigen is a protein, an antigenic determinant may be one of the many peptide sequences [generally 6-7 amino acids in length (M. Z. Atassi, Molec. Cell. Biochem. 32: 21-43 (1980)] within the entire protein molecule. Hence, monoclonal antibodies raised against a single antigen may be distinct from each other depending on the determinant that induced their formation; but for any given clone, all of the antibodies it produces are identical. Furthermore, the hybridoma cell line can be reproduced indefinitely, is easily propagated in vitro or in vivo, and yields monoclonal antibodies in extremely high concentration.
Monoclonal methods are generally applicable and have been used to produce antibodies to antigens other than the sheep red blood cells of Kohler and Milstein. For instance, it has been reported that monoclonal antibodies have been raised against tumor cells [U.S. Pat. No. 4,172,124], viruses [U.S. Pat. No. 4,196,265], and Group B Streptococci [R. Polin, Monoclonal antibodies against streptococcal antigen, pp. 353-359, in: R. Kennet, T. McKearn and K. Bechtol (editors), Monoclonal antibodies, hybridomas: a new dimension in biological analysis, Plenum Press, New York (1980)]. These antibodies are specific to each individual antigen against which they were raised and have no specificity for enterobacterial adhesins or enterobacterial strains bearing adhesins. Applicant believes that prior to this invention, no monoclonal antibodies have been produced that are specific to enterobacterial adhesin antigens involved in the colonization of enterotoxigenic bacteria in humans and animals. Furthermore, it is believed that no monoclonal antibodies have ever been utilized in the therapeutic or prophylactic treatment of diarrheal disease in humans or animals.